The long-term objectives of this application are to understand the molecular mechanisms of photoreceptor death in retinal degenerations and to search out new approaches to therapies for retinal degenerative disorders, such as retinitis pigmentosa and age-related macular degeneration. A large body of evidence indicates that caspases are central executioners of cell death. In many neuronal classes, caspase-3 is found to play an important role in the initiation of apoptosis. Preliminary studies suggest that a caspase-3-dependent mechanism is responsible for photoreceptor death in a line of transgenic rats bearing a rhodopsin mutation (S334ter) that exhibit rapid photoreceptor degeneration in the second week after birth. At issue is whether this caspase-3-dependent mechanism is common to most or all photoreceptor degenerations of different causes. If there is a common photoreceptor death mechanism, the way is open to design new treatments to target the key component(s) of this mechanism. This application focuses on the role of caspase-3 in photoreceptor degeneration in three degenerative models: the transgenic S334ter-3 rat, the rd mouse, and light-induced photoreceptor degeneration in the rat. Caspase-3 activation during degeneration will be characterized. Measurement of caspase-3 activity will be carried out using a fluorogenic substrate, Ac-DEVD-AMC. The amount of the p12 subunit of activated caspase-3 will be assessed by Western blotting. Consistent increases in caspase-3 activation during degeneration in all three models would strongly support the hypothesis of a common death mechanism. The role of caspase-3 in photoreceptor death will be further characterized using two different caspase inhibitors: an irreversible peptide caspase-3 inhibitor z-DEVD-fmk, and a baculovirus anti-apoptotic protein p35. The peptide inhibitor will be injected into the vitreous, and the p35 gene will be delivered to photoreceptors using a recombinant adenovirus vector. Protection of photoreceptors by these two inhibitors will indicate that caspase-3 is required for photoreceptor apoptosis. It will also provide the basis for preclinical studies of these and other caspase inhibitors for photoreceptor protection. In addition, neurotrophic factors that protect photoreceptors, such as CNTF and bFGF, will be tested for their effects on inhibiting caspase-3 activity. Results from these experiments will shed light on the mechanisms by which these neurotrophic factors protect photoreceptors. Finally, combined treatment with neurotrophic factors and caspase inhibitors will be used to investigate if enhanced protection can be achieved.